Application of MYLK and CPSF2 genes in regulating beef intramuscular fat deposition

Abstract

The present invention discloses the application of the MYLK gene and the CPSF2 gene in regulating beef intramuscular fat deposition, belonging to the field of genetic engineering technology. By studying the APA mechanism during cattle intramuscular fat deposition, the present invention found that during the IMF deposition process, the 3′UTR of the MYLK gene shortened and the expression level of the MYLK gene increased. During the shortening process, the miRNA involved in IMF deposition was lost, thereby regulating the reduction of IMF deposition; after the 3′UTR of the CPSF2 gene was shortened, its expression was also upregulated, regulating the reduction of IMF deposition. The research results of the present invention provide new theoretical support and technical guidance for the prediction of beef quality and the breeding of high-quality beef cattle, which is of great significance to promoting the development of animal husbandry and improving the production efficiency of animal husbandry.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the application of MYLK gene and CPSF2 gene in regulating beef intramuscular fat deposition. Background Art

[0002] In recent years, with the improvement of people's living standards, the demand for beef quality has become increasingly stringent. The process of intramuscular fat (IMF) deposition is crucial for the formation of meat texture and the improvement of taste. IMF content affects muscle shear force, flavor, juiciness, and tenderness. Flavor is closely related to fat and fat-soluble substances in muscle. As the main source of volatile compounds, IMF is considered a species-specific flavor precursor. The type and ratio of fatty acids in IMF significantly influence flavor compounds, and the flavor differences between different meats are due to their different oxidation products. The unsaturated fatty acids in IMF are essential fatty acids for the human body and have high nutritional value. IMF is present in the epimysium, perimysium, and endomysium of muscle fibers. IMF deposition promotes the separation of muscle fiber bundles, thereby improving muscle tenderness. Research by Zuo Xiuli et al. has shown that marbling grade has a highly significant positive impact on beef tenderness. Research by Mao Yanwei and others showed that IMF can increase the water-binding capacity of muscle, reduce drip loss and cooking loss of meat, and improve meat quality; in addition, IMF is negatively correlated with muscle shear force, but positively correlated with muscle pH value.

[0003] Factors influencing intramuscular fat deposition in beef cattle include genetics, management, and nutrition. IMF deposition can be regulated by specific genes (PPARγ, C / EBPα, ZFP423, and FABP4, among others) and signaling pathways (AMPK, WNT, and MAPK, among others). Furthermore, epigenetic modifications are also involved in the various biological processes of IMF deposition. However, in addition to transcriptional and epigenetic regulation, post-transcriptional regulation also plays a crucial role in IMF deposition.

[0004] Alternative polyadenylation (APA) is a post-transcriptional regulatory process that generates multiple transcripts with varying 3' untranslated region (3'UTR) lengths through selective splicing and the addition of polyadenylic acid tails of varying lengths. It plays a crucial role in gene expression regulation, influencing transcript stability, translation efficiency, and functional diversity. Current research indicates that variations in 3'UTR length can affect gene translation efficiency, and that the effects of APA on translation efficiency are primarily regulated by altering the availability of miRNA binding sites. APA is widely present in mammals and plays a key role in cellular development, biological processes, and disease progression.

[0005] Recent studies have revealed that APA is associated with IMF deposition and is involved in regulating muscle development and adipogenic differentiation. APA can mediate miRNA regulation of muscle stem cell function. Pax3, a key regulator of myogenesis, is affected by APA during myogenic satellite cell development, resulting in a shortened 3′UTR, making it resistant to miR-206 regulation. In mouse myoblasts, RBFOX2 regulates mRNA expression of contractile and mitochondrial genes through APA. Studies of the fast and slow skeletal muscle transcriptomes in porcine have shown that APA may play a role in fast and slow muscle development under the control of miRNAs and RNA-binding proteins. In 3T3-L1 preadipocytes, HO1 suppresses preadipocyte differentiation. APA-induced shortened HO1 3′UTR variants evade miRNA inhibition, resulting in higher HO1 expression and a stronger inhibitory effect on preadipocyte differentiation. However, studies on the mechanisms of APA during bovine IMF deposition are rare. Summary of the Invention

[0006] The present invention aims to address the aforementioned problems of the prior art by providing the use of the MYLK and CPSF2 genes in regulating intramuscular fat deposition in beef. By studying the APA mechanism during intramuscular fat deposition in cattle, the present invention discovered that the MYLK and CPSF2 genes regulate fat deposition, providing new theoretical support for predicting beef quality and breeding high-quality beef cattle.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of the MYLK gene in regulating beef intramuscular fat deposition.

[0009] The present invention also provides application of the CPSF2 gene in regulating beef intramuscular fat deposition.

[0010] The present invention also provides application of a reagent for detecting the expression level of the MYLK gene in predicting or assisting in predicting beef quality.

[0011] The present invention also provides application of a reagent for detecting the expression amount of the MYLK gene in breeding beef cattle with high-quality beef.

[0012] The present invention also provides the use of a reagent for detecting the expression level of the CPSF2 gene in predicting or assisting in predicting beef quality.

[0013] The present invention also provides application of a reagent for detecting the expression level of the CPSF2 gene in breeding beef cattle with high-quality beef.

[0014] The present invention discloses the following technical effects:

[0015] This study investigated the APA mechanism during intramuscular fat deposition in cattle and established an APA profile during IMF deposition. The findings indicate that 3′UTR shortening is associated with IMF deposition, which can lead to increased gene expression levels through the loss of miRNA binding sites. Further research revealed that during IMF deposition, the 3′UTR of the MYLK gene shortens, leading to increased MYLK gene expression. During this shortening process, bta-miR-206, bta-miR-32, and bta-miR-195, which are involved in IMF deposition, are lost, regulating the reduction of IMF deposition. Shortening of the 3′UTR of the CPSF2 gene also results in increased expression, regulating the reduction of IMF deposition. The findings provide new theoretical support and technical guidance for predicting beef quality and breeding high-quality beef cattle, and are of great significance for promoting the development of animal husbandry and improving its production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The results of the comprehensive analysis of RNA-seq data; A is the PDUI score plot for each gene in groups H and L, the dotted line indicates the threshold of 0.1, blue dots represent genes with extended 3'UTRs, and red dots represent genes with shortened 3'UTRs; B is the volcano plot of A, representing |δPDUI|>0.1 (FDR<0.05) for genes with shortened (red) and extended (blue) 3'UTRs; C is a comparison of the 3'UTR lengths of transcripts that were significantly changed in group H and other transcripts that did not exceed the threshold detected in samples from groups H and L; D is a bar graph of the number of base pairs lost / gained by genes with altered 3'UTRs;

[0018] Figure 2 is the relationship between 3′UTR length and aUTR length;

[0019] Figure 3 Figure 2 shows the detection results of related gene expression in APA-driven IMF deposition; A is a graph showing the log2-fold change in expression of genes with 3'UTR alterations; the upregulated genes (red dots) and downregulated genes (blue dots) on the left represent genes with shortened 3'UTRs, while the right represents genes with extended 3'UTRs; B is a comparison of the number of genes with upregulated (red) or downregulated (blue) 3'UTR alterations in IMFs;

[0020] Figure 4 Figure 3 shows the establishment and analysis of the miRNA-APA network during fat deposition. Figure A shows the distribution of miRNA binding sites lost due to 3'UTR shortening. Figure B shows the miRNA-APA network, with lines representing miRNAs affected by APA genes and gene-gene interactions, with PPI > 0.7. Figure C shows the regulatory pattern of APA and MYLK 3'UTRs with conserved miRNA site locations.

[0021] Figure 5 is the expression of MYLK gene in group H and group L;

[0022] Figure 6 Figure 2 is the expression of CPSF2 gene in group H and group L. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Method

[0030] 1.1 Sample collection and IMF content determination

[0031] Thirty crossbred Wagyu cattle (Qinchuan cattle × Wagyu cattle) in good health, of similar age and weight were selected and housed together. These cattle were fed under the same nutritional level and management conditions. Before slaughter, the cattle needed to fast for 24 hours, but had free access to water. After slaughter according to standard procedures, the longissimus dorsi muscle was collected and frozen in liquid nitrogen, and then stored at -80°C for RNA extraction. At the same time, approximately 500g of the longissimus dorsi muscle was collected for determination of IMF content. The IMF content of the longissimus dorsi muscle was determined by Soxhlet extraction. From the 30 cattle, three individuals with high IMF content (H) (32.96±3.26%) and three individuals with low IMF content (L) (10.91±1.85%) were selected for further analysis. The IMF content of these individuals was significantly different (P<0.05).

[0032] 1.2 RNA preparation, library construction, and sequencing

[0033] Total RNA was extracted using a Trizol kit (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase-free agarose gel electrophoresis. After total RNA extraction, rRNA was removed, and mRNA and ncRNAs were retained. The enriched mRNAs and ncRNAs were fragmented into short fragments using a fragmentation buffer and reverse transcribed into cDNA using random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified using a QiaQuick PCR extraction kit (Qiagen, Venlo, the Netherlands), the ends were repaired, poly (A) was added, and the fragments were ligated to Illumina sequencing adapters. The second-strand cDNA was then digested with UNG (uracil-N-glycosylase). The digested products were size-selected by agarose gel electrophoresis, PCR amplified, and sequenced using an Illumina HiSeq™ 4000.

[0034] 1.3APA analysis

[0035] Sequence files were quality controlled using FastQC v0.11.9 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and low-quality reads were trimmed using Trim Galore v0.6.0 (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). All sequence files were aligned to the bovine reference genome ARS-UCD1.2 using HISAT2 v2.2.1. BAM files for the six samples were converted to wig files using bedtoolsgenomecov v2.30.0. The wig files were processed using DaPars v1.0.0 (https: / / github.com / ZhengXia / DaPars) to identify differences in 3′ UTRs between samples with high and low IMF content. Proximal poly A sites were predicted, and the percentage of distal poly A site usage index (PDUI) was calculated. PDUI values ​​range from 0 to 1. Higher PDUI scores indicate higher utilization of distal PAS sites.

[0036] To compare 3′UTR changes in specific genes between high-IMF and low-IMF samples, the PDUI scores for high-IMF samples (MeanPDUIH) and low-IMF samples (MeanPDUIL) were averaged. For each gene, the mean change in PDUI score between high-IMF and low-IMF samples (ΔPDUI = MeanPDUIH - MeanPDUIL) was calculated as a measure of 3′UTR differences. The significance of this difference was assessed using the Fisher's exact test and further adjusted using the Benjamini-Hochberg (BH) test to control the false discovery rate, using a threshold of 0.05. Genes with significant changes in the 3′UTR landscape (padj ≤ 0.05) were retrieved and then subjected to gene enrichment analysis using DAVID v6.8 (https: / / david.ncifcrf.gov / ).

[0037] 1.4 Differential gene expression analysis

[0038] StringTie v2.1.7 was used to generate raw count matrices for the six samples and converted to transcripts per million (TPM) matrices. Differentially expressed genes (DEGs) were detected using the R package DESeq2 v1.20. The threshold for screening DEGs was |log2FC| ≥ 0.585. The p-value was corrected for FDR using Benjamini-Hochberg correction, with an FDR < 0.05 as the threshold.

[0039] 1.5 miRNA-APA network construction

[0040] The raw count matrix of miRNA expression for the six samples was normalized to transcripts per million (TPM), and miRNAs with an average TPM ≥ 100 were defined as highly expressed miRNAs. MiRNA binding site information was downloaded from TargetScanHuman 7.2, and the binding sites of highly expressed miRNAs were mapped in the aUTR regions of differential APA events. PPI network analysis of genes associated with differential APA events was performed using STRING v11.5, and gene pairs with interaction scores greater than 0.7 were retained. Finally, miRNA-APA was merged with PPI, and the miRNA-APA network was visualized using Cytoscape v3.10.0.

[0041] 1.6Integrated genomics viewer

[0042] IGV v2.14.0 was used to display the length and abundance of 3′UTR sequences in samples with high and low IMF content. The reference genome (Bos_taurus.ARS-UCD1.2.dna.toplevel.fa) and annotation file (Bos_taurus.ARS-UCD1.2.107.gtf) were downloaded from the Ensembl database and imported into IGV. Bam files for the six samples were then imported into IGV to examine the length and sequence differences of the 3′UTRs.

[0043] 2. Results

[0044] 2.1 APA events during fat deposition

[0045] To explore the changes in APA during bovine IMF deposition, RNA-seq data from six samples (three longissimus dorsi muscles with high and three longissimus dorsi muscles with low IMF content) were analyzed by DaPars. To compare the 3′UTR changes of a gene between samples with high IMF content (H) and low IMF content (L), the PDUI score of the gene was averaged in samples with high IMF content (MeanPDUIH) and low IMF content (MeanPDUIL). The change in the average PDUI score of each gene between H and L samples was calculated (ΔPDUI=MeanPDUIH-MeanPDUIL) and used as a measure of 3′UTR shortening or lengthening events associated with fat deposition. After strict screening (adjusted p-value<0.05; |MeanPDUIH-MeanPDUIL|>0.1, Figure 1 A and B), 363 differential APA events were obtained, and the 3'UTRs of 300 genes were determined to be significantly different. Between the H and L longissimus dorsi muscles, 175 genes showed 3'UTR shortening (ΔPDUI=MeanPDUIH-MeanPDUIL≥0.1), while 125 genes showed 3'UTR extension (ΔPDUI=MeanPDUIH-MeanPDUIL<-0.1). It is worth noting that the number of shortened 3'UTR events exceeded the number of extended 3'UTR events. Transcripts for which significant changes were observed showed longer 3'UTR lengths than transcripts below the threshold ( Figure 1 The lengths of IMF deposition-related shortening and lengthening events were mainly 200-300 bp and >500 bp, respectively ( Figure 1 The variable 3′UTR (aUTR) length was defined as the distance between the distal polyA site and the proximal polyA site. The results showed that the aUTR length of most APA events was 200-300 nt. At the same time, as the 3′UTR lengthened, the aUTR also lengthened (R 2 =0.79)( Figure 2 These findings suggest that genes with longer 3′UTRs are more likely to be regulated by APAs during IMF deposition.

[0046] 2.2 APA-driven changes in gene expression

[0047] To determine whether APA events drive changes in gene expression in fat deposition, we calculated differential gene expression between tissues with high and low IMF content (H and L) and investigated the association between specific APA events and gene expression changes ( Figure 3Among genes with shortened 3′UTRs, 16 genes were significantly upregulated and 18 genes were significantly downregulated. Similarly, among genes with extended 3′UTRs, 10 genes were significantly upregulated and 11 genes were significantly downregulated ( Figure 3 (B) The increased expression of 16 genes may be the result of genes with shortened 3′UTRs escaping miRNA repression. However, compared with all DEGs, the upregulation of genes with shortened 3′UTRs was not significant, suggesting the presence of other regulatory mechanisms for gene expression. Furthermore, three genes with extended 3′UTRs, ACVR2B, LMOD3, and RTN4, were upregulated during IMF deposition. These three genes are associated with myocyte proliferation and differentiation. Previous studies have shown that 3′UTR variants of some genes can increase their expression through trans-regulatory mechanisms.

[0048] 2.3 miRNA-APA network in fat deposition

[0049] To verify that 3′UTR shortened by APA during IMF deposition may escape miRNA inhibition and thus increase gene expression, the distribution of miRNA binding sites lost due to 3′UTR shortening was calculated. This analysis showed that 44.1% of genes lost at least one highly conserved miRNA binding site ( Figure 4 A), indicating that the alteration of miRNA binding sites is a common regulatory mode mediated by APA.

[0050] To further analyze the changes in miRNA binding sites that may be caused by APA events during IMF deposition, miRNA expression data of 6 samples were collected, and miRNA expression was normalized using TPM values. Highly expressed miRNAs with TPM ≥ 100 were selected, and then these miRNA binding sites were aligned to the aUTRs of differential APA events. In this way, a total of 146 important IMF-related miRNA sites were found that may be gained or lost due to 363 differential APA events. Afterwards, STRING was used to identify protein-protein interactions (PPIs) of APA events. These results were then used to construct a miRNA-APA network ( Figure 4 B), among the top 10 miRNAs regulating this network, bta-miR-32 has been shown to regulate fat deposition. Bta-miR-206, bta-miR-195, and bta-miR-16a have been reported to be associated with the proliferation and differentiation of muscle cells, so they may also be involved in regulating the deposition of IMF. In this regulatory network, an important gene, MYLK, was also found. When the 3′UTR of MYLK is shortened, the miRNA binding sites of bta-miR-32, bta-miR-206, and bta-miR-195 are lost ( Figure 4Therefore, MYLK may be a key gene in the IMF deposition process.

[0051] Furthermore, a key regulator of APA events, CPSF2, has been discovered. During IMF deposition, CPSF2 gene expression is upregulated following 3′UTR shortening. Cleavage and Polyadenylation Specificity Factor Subunit 2 (CPSF2), a member of the cytoplasmic polyadenylation enzyme (CPF) family, is a gene that recognizes polyadenylation signals. CPSF2 recognizes the AAUAAA polyadenylation signal and plays an important role in nuclear export, translation initiation, and transcript stability. CPSF2 may be a target of intronic miRNAs. When CPSF2 is silenced, polyadenylation is biased toward recognizing the typical poly(A) signal, resulting in elongated 3′UTRs for most genes. In cancer cells, CPSF2 modulates APA events in glycolysis-related genes, shortening their 3′UTRs and promoting cancer cell growth by upregulating the glycolytic pathway. Furthermore, during cancer cell development, increased expression of mRNA 3′-end processing factors, including CPSF2, leads to shortened mRNA 3′UTRs. Therefore, it is believed that CPSF2 may be a key regulatory factor in the process of IMF deposition. The miRNA-APA regulatory network of MYLK and CPSF2 is shown in Table 1.

[0052] Table 1 miRNA-APA regulatory network constructed using STRING and TargetScanHuman

[0053]

[0054]

[0055] Example 2 Differential expression of MYLK and CPSF2 genes

[0056] 1. Design and synthesis of primers

[0057] Primers were designed using Primer Premier 5 software based on the cDNA sequences of bovine MYLK, CPSF2, and GAPDH genes in Genebank. The primer sequences for each gene are shown in Table 2.

[0058] Table 2 Primer sequences

[0059]

[0060] 2. RNA Extraction

[0061] (1) The bovine intramuscular adipose tissue of the individuals in Group H and Group L determined in Example 1 was removed from a -80°C freezer and placed on ice. After it was completely dissolved, the tissue block was removed from the RNAsolid tissue RNA stabilization solution using clean tweezers. The surface liquid was then absorbed with clean absorbent paper and placed in a 1.5 ml EP tube.

[0062] (2) Add 1 ml of lysis buffer RZ to every 50-100 mg of tissue and homogenize using a homogenizer.

[0063] (3) Place the homogenized sample at 15-30°C for 5 minutes to allow the nucleic acid-protein complex to be completely separated.

[0064] (4) Centrifuge at 4°C, 12,000 rpm for 5 min, and transfer the supernatant into an RNase-free centrifuge tube.

[0065] (5) Add 200 μL of chloroform and tightly cap the centrifuge column. Shake vigorously for 15 seconds and let stand at room temperature for 3 minutes.

[0066] (6) Centrifuge at 12,000 rpm at 4°C. The sample in the centrifuge column will separate into an upper colorless aqueous phase containing RNA, an intermediate phase, and a lower yellow organic phase. The volume of the aqueous phase is approximately half the volume of the RZ. Use a pipette to carefully transfer the upper aqueous phase to a new centrifuge tube.

[0067] (7) Add 200 μL of anhydrous ethanol and shake to mix thoroughly. Transfer the resulting precipitate and liquid to adsorption column CR3. Centrifuge at 12,000 rpm for 30 s at 4°C, and discard the waste liquid in the collection tube below the adsorption column.

[0068] (8) Add 500 μL of deproteinized solution RD to the adsorption column CR3. Centrifuge at 4°C, 12,000 rpm for 1 min. Discard the waste liquid in the collection tube.

[0069] (9) Add 500 μL of rinse solution RW to the adsorption column CR3 and let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 30 s at 4°C and discard the waste liquid in the collection tube.

[0070] (10) Repeat step (9) to ensure that the impurities are completely rinsed.

[0071] (11) Transfer the adsorption column CR3 to a new 1.5 mL centrifuge tube and add 30 μL of RNase-Free ddH 2 O. Let it stand at room temperature for 5 min and then centrifuge at 12,000 rpm for 2 min at 4°C.

[0072] (12) Use Nanodrop to measure RNA concentration and control OD 260 / OD 280 Approximately equal to 2.

[0073] (13) RNA gel electrophoresis was used to verify RNA integrity.

[0074] 3. cDNA Preparation

[0075] Follow the manufacturer's instructions for the reverse transcription kit. Using eight-well plates, add 4 μl of 5× PrimeScript RT Master Mix and 1000 ng of RNA to each well, adjusting the volume to 20 μl with DEPC water. Mix thoroughly using a microfuge. Place in a PCR instrument and reverse transcribe at 37°C for 15 min. Heat inactivate the reverse transcriptase at 85°C for 5 seconds, cool to 4°C, and store at -20°C until needed.

[0076] 4. qRT-PCR reaction

[0077] The specific operation steps of qRT-PCR and the amount of each reagent added are shown in Table 3.

[0078] Table 3 Reagents and dosages required for qRT-PCR

[0079]

[0080]

[0081] Add the prepared reaction solutions of each dose group to eight tubes, mix and centrifuge, and place in a fluorescence quantitative PCR instrument. The reaction setting program is shown in Table 4.

[0082] Table 4 PCR reaction program

[0083]

[0084] GAPDH gene was used as internal reference, and the relative content of target gene amplification product was expressed as 2 -ΔΔCt The specific calculation method is as follows: First, the Ct value of the target gene is minus the Ct value of the internal reference gene: ΔCT = CT (target gene) – CT (internal reference); Second, the ΔCt value of the treated sample is minus the ΔCt value of the control sample: ΔΔCT = ΔCT (experimental sample) – ΔCT (control sample). Any sample that can represent a fold increase in the expression level of the target gene is used as the control sample; Finally, the expression level ratio is calculated: 2 -ΔΔCt = ratio of relative gene expression levels.

[0085] The expression of MYLK and CPSF2 genes in group H and group L is shown in Figure 2. Figure 5 and Figure 6 As shown in the figure, it can be seen that the expression of MYLK and CPSF2 genes in the two groups was significantly different. Combined with the above results, MYLK and CPSF2 genes are important genes regulating IMF deposition.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of a reagent for detecting MYLK gene expression in predicting beef quality, characterized in that: The beef quality mentioned refers to the intramuscular fat content.

2. The use of a reagent for detecting the expression level of the MYLK gene in breeding beef cattle with high-quality beef, characterized in that: The beef quality mentioned refers to the intramuscular fat content.

3. Application of a reagent for detecting CPSF2 gene expression in predicting beef quality, characterized in that: The beef quality mentioned refers to the intramuscular fat content.

4. Use of a reagent for detecting CPSF2 gene expression in breeding beef cattle with high-quality beef, characterized in that: The beef quality mentioned refers to the intramuscular fat content.

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